This invention relates to heparin cofactor II and, more particularly, to a modified heparin cofactor II that has substantially improved inhibitory activity against thrombin compared to native heparin cofactor II or antithrombin.
The anticoagulant activities of glycosaminoglycans are mediated by antithrombin (AT) and heparin cofactor II (HCII), which are members of the `serpin` family of serine protease inhibitors [Tollefsen in Heparin: chemical and biological properties, clinical applications (eds. Lane and Lindahl), Edward Arnold, London, 1989, pp. 257-273]. HCII inhibits thrombin but has no activity against other proteases involved in coagulation or fibrinolysis [Parker and Tollefsen, J. Biol. Chem. 260, 3501-3505 (1985)]. In contrast, AT inhibits all of the proteases of the intrinsic coagulation pathway as well as the fibrinolytic protease plasmin [Rosenberg in The molecular basis of blood diseases (eds. Stamatoyannopoulos et al.), W. B. Saunders Co., Philadelphia, 1987, pp. 534-574]. Heparin and dermatan sulfate increase the rate of thrombin inhibition by HCII.about.1000-fold by providing a catalytic template to which both the inhibitor and the protease bind [Tollefsen et al., J. Biol. Chem. 258, 6713-6716 (1983); Griffith, Proc. Natl. Acad. Sci. USA 80, 5460-5464 (1983)]. Although heparin catalyzes protease inhibition by both HCII and AT, the anticoagulant effect of dermatan sulfate is mediated exclusively by HCII.
HCII is a 65,600 dalton plasma glycoprotein. Using conventional recombinant DNA gene splicing methods, the cDNA sequence and corresponding amino acid sequence of HCII were determined and the protein was expressed in E. coli by Blinder et al., Biochemistry 27, 752-759 (1988). The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a native protein of 480 amino acids. The published cDNA and protein sequences are also shown hereinbelow.
HCII forms a stable 1:1 complex with thrombin or chymotrypsin in which the protease is inactive. During complex formation, both thrombin and chymotrypsin attack the reactive site Leu.sub.444 -Ser.sub.445 peptide bond (designated P1-P1') near the C-terminal end of HCII [Church et al., Proc. Natl. Acad. Sci. USA 82, 6431-6434 (1985); Griffith et al., J. Biol. Chem. 260, 2218-2225 (1985); Griffith et al., Biochemistry 24, 6777-6782 (1985)]. The resulting complex does not dissociate when heated at 100.degree. C. with sodium dodecyl sulfate, suggesting that under these conditions the two proteins become linked by a covalent bond. The bond is presumed to be an ester linkage between the serine hydroxyl group in the active site of the protease and the carbonyl group of Leu.sub.444 in HCII. Therefore, HCII can be thought of as a pseudosubstrate for these proteases.
The P1 residue in the reactive site of a `serpin` appears to play a major role in determining the relative rates of inhibition of various proteases [Carrell and Travis, TIBS 10, 20-24 (1985)]. Evidence for this was derived from analysis of the variant .alpha.1-antitrypsin Pittsburgh, which was discovered in a child who had a fatal bleeding disorder. The variant contained an arginine residue in place of Met.sub.358 at the P1 position of the inhibitor [Owen et al., N. Engl. J. Med. 309, 694-698 (1983)]. This substitution resulted in markedly increased rates of inhibition of several coagulation proteases, including thrombin, factor Xa, factor XIa, and kallikrein, which cleave Arg-X peptide bonds in their natural substrates [Schapira et al., J. Clin. Invest. 77, 635-637 (1986); Scott et al., Ibid. 77, 631-634 (1986); Travis et al., Biol. Chem. Hoppe-Seyler 367, 853-859 (1986)]. A reciprocal decrease in the rate of inhibition of neutrophil elastase, the principal target protease of native .alpha.1-antitrypsin, was also noted.
Heparin is commonly employed in the prophylaxis and treatment of venous thrombosis and pulmonary embolism, but its use is sometimes complicated by severe bleeding or thrombocytopenia. Therefore, alternatives to heparin for short-term anticoagulation would be desirable. In this regard, a modified HCII which could inhibit thrombin rapidly in the absence of heparin would have significant value and be useful for the treatment of thrombotic disorders.